Template-switching of CMV-RNA at replication.

CMV-RNA 在复制时的模板转换。

• 批准号: 10660044
• 负责人: OKUDA Seiichi
• 金额: $ 2.18万
• 依托单位: Utsunomiya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660044/
• 关键词: Plant Virus; Satellite RNA; Virus Replication; Template Switching; テンペレートスイッチング; キュウリモザイクウイルス; タバコモザイクウイルス; ウイルスRNA

Previously we have showed that multimers of satellite RNA are present in cucumber mosaic virus (CMV) infecting tobacco leaves. First of all in this research we assumed that the hetero-joined RNA molecules were synthesized in virus-infected plants, for example CMV satellite RNA joined with CMV genomic RNA, oligonucleotide primers were designed on the basis of the nucleotide sequence of CMV genome- and satellite RNAs to amplify the junction region in head-to-tail hetero-joined molecules. The cDNA was synthesized from total RNA extracted from CMV-infecting tobacco leaves and amplified by PCR with the primers. Sequence analysis of RT-PCR products revealed that they were plus-sense or minus-sense head-to-tail joined RNAs between CMV genomic RNA1-3 but not plus-minus joined one. The junction sequences indicated that most molecules lacked sequence of 3' components and a few have insertion between 5' and 3' components. However, no head-to-tail joined RNA was detected from purified CMV preparation. We have also detected plus-sense or minus-sense head-to-tail joined RNAs from samples infected with cucumber green mottle mosaic virus (CGMMV). These data indicate that hetero-joined vital RNA molecules are generated by a replicase-driven copy-choice mechanism. In this research we have succeeded to develop the simple direct tube RT-PCR method that can detect virus RNA.Moreover, to confirm the head-to-tail joined RNAs from virus-infecting samples, the infectious full-length cDNA clone of CMV was constructed.

先前我们已经证实，在侵染烟叶的黄瓜花叶病毒（CMV）中存在卫星RNA多聚体。本研究首先假设在病毒感染的植物中合成了异连接RNA分子，如CMV卫星RNA与CMV基因组RNA连接，根据CMV基因组RNA和卫星RNA的核苷酸序列设计寡核苷酸引物，扩增头尾异连接分子的连接区域。从cmv侵染烟叶中提取总RNA合成cDNA，用引物进行PCR扩增。RT-PCR产物序列分析显示，在CMV基因组RNA1-3之间存在正义或负义首尾连接rna，而非正负连接rna。连接序列表明，大多数分子缺乏3′组分序列，少数分子在5′和3′组分之间插入。然而，从纯化的CMV制备中未检测到头尾连接RNA。我们还从黄瓜绿色斑驳花叶病毒（CGMMV）感染的样品中检测到正义或负义从头到尾连接的rna。这些数据表明，异连接的重要RNA分子是由复制酶驱动的复制选择机制产生的。本研究成功地建立了一种简单的直接管RT-PCR检测病毒RNA的方法。此外，为了确认病毒感染样本的头尾连接rna，构建了CMV感染性全长cDNA克隆。